Collagen is the principle load-bearing molecule and most abundant protein in the body. Collagen degenerative diseases include osteoarthritis (affecting 10% of Americans 60 years or older), osteoporosis (affecting 28 million Americans), arterial aneurysms and intervetebral disc disease. There are 50,000 corneal transplants and 200,000 primary ACL reconstructions performed in the US each year demonstrating the need for suitable, mechanically strong graft material. Collagen-related genetic disorders are equally devastating; examples include osteogenesis imperfecta (affecting 27,000 people in the US), many chondrodysplasias (1 in 4,000 births), and various syndromes (i.e. Ehlers-Danlos, Alport and Bethlem myopathy). The economic impact of these diseases is enormous; osteoarthritis alone costs the US economy more than $60 billion annually. In spite of this, little is known about the mechanisms that govern collagen assembly in vivo which has limited the ability of tissue engineers to reproduce collagenous load-bearing tissue in the laboratory. Instead, tissue engineering utilizes cell-seeded, degradable synthetic or self-assembled, collagenous scaffolds which require resorption and remodeling to be functional. Resorption and remodeling of scaffolds closely resembles in vivo scar remodeling or wound healing which can take years to complete, even under ideal conditions. Consequently, no functional load-bearing tissue-engineered equivalents have met with clinical success. A method to construct mechanically strong, collagenous matrix should improve the chances of producing functional connective tissue equivalents. Collagen matrices must be highly ordered and highly concentrated to possess adequate strength. It is thus necessary to organize collagen at the nanoscale in a manner similar to embryonic development where fibroblast cells exercise exquisite control over the fibrillogenesis of collagen. Though little is known about the detailed mechanics of this process there are two competing hypotheses which suggest that collagen is either: a) synthesized, assembled and organized by individual cells via cell-surface fibropositors or b) secreted in monomer form and then assembled into an organized matrix via cholesteric effects. In the proposed work, we will observe organized matrix assembly using time-lapse live dynamic differential interference contrast imaging of individual cells in a novel in vitro model of corneal stromal ECM development. We will then produce organized collagenous matrices de novo by mimicking the development mechanism in one of two in vitro cell-free systems currently under development in our laboratory. If successful, the proposed investigation will answer a critical basic science question regarding development and then translate those results into a process capable of producing load-bearing collagenous tissue equivalents. [unreadable] [unreadable] [unreadable]